Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.     

Where am I and why? Synthesizing range biology and the eco‐evolutionary dynamics of dispersal

Although generations of researchers have studied the factors that limit the distributions of species, we still do not seem to understand this phenomenon comprehensively. Traditionally, species’ ranges have been seen as the consequence of abiotic conditions and local adaptation to the environment. However, during the last years it has become more and more evident that biotic factors – such as intra‐ and interspecific interactions or the dispersal capacity of species – and even rapidly occurring evolutionary processes can strongly influence the range of a species and its potential to spread to new habitats. Relevant eco‐evolutionary forces can be found at all hierarchical levels: from landscapes to communities via populations, individuals and genes. We here use the metapopulation concept to develop a framework that allows us to synthesize this broad spectrum of different factors. Since species’ ranges are the result of a dynamic equilibrium of colonization and local extinction events, the importance of dispersal is immediately clear. We highlight the complex interrelations and feedbacks between ecological and evolutionary forces that shape dispersal and result in non‐trivial and partially counter‐intuitive range dynamics. Our concept synthesizes current knowledge on range biology and the eco‐evolutionary dynamics of dispersal. Synthesis What factors are responsible for the dynamics of species' ranges? Answering this question has never been more important than today, in the light of rapid environmental changes. Surprisingly, the ecological and evolutionary dynamics of dispersal – which represent the driving forces behind range formation – have rarely been considered in this context. We here present a framework that closes this gap. Dispersal evolution may be responsible for highly complex and non‐trivial range dynamics. In order to understand these, and possibly provide projections of future range positions, it is crucial to take the ecological and evolutionary dynamics of dispersal into account.     

Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of beta-arrestin in the resensitization of the beta(2) adrenergic receptor (beta(2)AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit beta-arrestin to the plasma membrane and internalize in a beta-arrestin- and clathrin-dependent manner. However, whereas beta-arrestin dissociates from the beta(2)AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of beta-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of beta-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of beta-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which beta-arrestins regulate the reestablishment of GPCR responsiveness.     

Genetic models with reduced penetrance related to the Y chromosome

Classical statistical genetics models of a quantitative trait depending on an autosomal gene indicate that father-to-daughter and mother-to-son correlations should be the same. If phenotypes are not sex-dependent, father-to-son and mother-to-daughter correlations also share this common value. On the other hand, if the gene is sex-linked, then the father-to-son correlation is zero. Such models do not explain genetic variation in pulmonary artery pressure (PAP) of cattle--important because cattle with high PAP are known to develop brisket disease, pulmonary heart disease, and congestive heart failure when taken to high altitudes. Data on 966 calves at a ranch in Colorado showed positive correlation (0.2) between sire PAP and male calf PAP but slightly negative correlation (-0.01) between sire PAP and female calf PAP; the dam-to-male calf and dam-to-female calf correlations are both about 0.1. The model presented here postulates an autosomal gene with reduced penetrance (i.e., the trait may remain at a normal level even when the genotype suggests abnormality) and that, in males, the rate of penetrance is related to an abnormality in the Y chromosome and is therefore passed on from father to son. Then under plausible selective breeding assumptions, the pairwise correlation between fathers and daughters can become zero or negative. Explicit formulas are computed for the model covariances, and numerical computations indicate that plausible parameter values can be chosen for the model.     

The track structures of ionizing particles and their application to radiation biophysics

A new approach to the interpretation of the effects of radiation on cells is described, in which sample particle tracks are constructed using a Monte Carlo computer program and the exposure of cellular targets to these tracks is simulated using a second program known as BIOPHYS. Data on the shapes and DNA contents of the cell nuclei are obtained from the literature. It is assumed that the sensitive material is DNA, and that the target is divided into cubes of approximately 2 nm (the diameter of the DNA helix) per side; the numbers of these cubes containing different numbers of ionizations are derived. Two different methods of analysing the output of BIOPHYS are described. In the first, it is assumed that lethality is caused by the occurrence of a number of ionizations equal to or greater than a certain threshold in one cube; in the second method, it is assumed that only two ionizations are required, in different parts of the cube, but that only some fraction of the cube is sensitive. These models have been applied to the interpretation of the variation of radiosensitivity with a linear energy transfer (LET) of spores of Bacillus subtilis exposed wet and dry, and good fits to the published experimental data were obtained using both models. Fits to experimental data for a range of other cell lines will be presented in a second paper. Received: 5 August 1998 / Accepted in revised form: 28 April 1999     

Genetic Manipulation of Alcohol Dehydrogenase Levels in Ripening Tomato Fruit Affects the Balance of Some Flavor Aldehydes and Alcohols

Tomato (Lycopersicon esculentum) plants were transformed with gene constructs containing a tomato alcohol dehydrogenase (ADH) cDNA (ADH 2) coupled in a sense orientation with either the constitutive cauliflower mosaic virus 35S promoter or the fruit-specific tomato polygalacturonase promoter. Ripening fruit from plants transformed with the constitutively expressed transgene(s) had a range of ADH activities; some plants had no detectable activity, whereas others had significantly higher ADH activity, up to twice that of controls. Transformed plants with fruit-specific expression of the transgene(s) also displayed a range of enhanced ADH activities in the ripening fruit, but no suppression was observed. Modified ADH levels in the ripening fruit influenced the balance between some of the aldehydes and the corresponding alcohols associated with flavor production. Hexanol and Z-3-hexenol levels were increased in fruit with increased ADH activity and reduced in fruit with low ADH activity. Concentrations of the respective aldehydes were generally unaltered. The phenotypes of modified fruit ADH activity and volatile abundance were transmitted to second-generation plants in accordance with the patterns of inheritance of the transgenes. In a preliminary taste trial, fruit with elevated ADH activity and higher levels of alcohols were identified as having a more intense “ripe fruit” flavor.     

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters

A neutral protease with an estimated M_r of about 26 kD and responsible for cleavage of myosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.     

Brown‐midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene

Brown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.